Multiplex Immunoassay for Serological Diagnosis of Mycobacterium bovis Infection in Cattle

Enfer Scientific, Unit T, M7 Business Park, Newhall, Naas, County Kildare, Ireland.
Clinical and vaccine Immunology: CVI (Impact Factor: 2.47). 11/2008; 15(12):1834-8. DOI: 10.1128/CVI.00238-08
Source: PubMed


Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.

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    • "For these reasons, the detection of Abs continues being a bottleneck and it has not played an important role in eradication programmes yet since it is not included as an official diagnostic tool. A variety of ELISA tests have been developed that rely on the detection of circulating Abs against the immunodominant antigens of M. bovis (Green et al., 2009; Waters et al., 2011; Whelan et al., 2008). In general, these assays are simple, rapid and inexpensive although they have shown lower Se than the assays based on cell mediated immune response. "
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    ABSTRACT: Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations.
    Full-text · Article · Oct 2014 · Research in Veterinary Science
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    • "Waters et al., 2011 tested MPB83 and MPB70 using sera from infected cattle and reported apparent sensitivity and specificity of 63% and 98%, respectively [16]. ESAT-6 and CFP-10 were used in an ELISA format by Whelan et al., 2008 using a total of 1489 bovine TB negative and 522 bovine TB positive cattle sera and stated sensitivity of 40.6% - 86.6% and specificity of 82.6% - 69.7%, based upon use of two different test interpretation criteria [26]. In the present study, we detected anti- M. bovis antibodies in 77.2% and 87.5% infected cattle and deer, respectively. "
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    ABSTRACT: Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. Methods By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. Results Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. Conclusion The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.
    Full-text · Article · Jul 2014 · BMC Veterinary Research
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    • "Thus, chimeras containing important epitopes of major proteins of M. bovis are expected to be able to detect antibodies from cattle in different stages of infection, thereby increasing the diagnostic coverage. Similar results are reported in a chemiluminescent assay with multiple antigens , in which sensitivity and specificity were superior to those of individual antigens, including ESAT-6 and MPB83 (Whelan et al. 2008). The choice for the production of a chimera of antigens instead of a cocktail of individual antigens is justified by the facility of a single expression and "
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    ABSTRACT: Bovine tuberculosis is an important infectious disease caused by Mycobacterium bovis, which is responsible for considerable economic losses. This disease constitutes a serious public health problem. Control programs in most countries, including Brazil, are based on the identification and slaughter of infected animals, as defined by the skin tuberculin test, which has its constraints. In the present study, the recombinant proteins CFP-10, ESAT-6, Mb0143, MPB83, PE5, PE13, TB10.4, TB15.3 and a chimera of ESAT-6/MPB70/MPB83 (fusion protein) were tested as ELISA antigens for the diagnosis of bovine tuberculosis. The proteins were produced in Escherichia coli, purified and tested in ELISAs with sera from 126 cattle having tested negative in the comparative intradermal tuberculin test (CITT) and 107 sera from cattle having tested positive in the CITT. Also, 236 sera from two BTB-free beef cattle herds were tested. Among the proteins tested, only the ESAT-6/MPB70/MPB83 chimera demonstrated satisfactory agreement with the CITT (kappa index: 0.688), reflecting in 83.2% sensitivity and 86.5% specificity. The ELISA absorbances of the cattle sera from BTB-free herds showed similar levels to those of CITT positive cattle, probably as the result of successive skin tuberculinizations to define the BTB-free status of the herds. However, the ELISA with the ESAT-6/MPB70/MPB83 chimera was useful to discriminate BTB positive and negative cattle in herds prior to the tuberculin skin test.
    Full-text · Article · Dec 2012 · SpringerPlus
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